Abstract
The serial crystallography (SX) technique enables the determination of the room-temperature structure of a macromolecule while causing minimal radiation damage, as well as the visualization of the molecular dynamics by time-resolved studies. The fixed-target (FT) scanning approach is one method for SX sample delivery that minimizes sample consumption and minimizes physical damage to crystals during data collection. Settling of the crystals on the sample holder in random orientation is important for complete three dimensional data collection. To increase the random orientation of crystals on the sample holder, we developed a polyimide mesh-based sample holder with irregular crystal mounting holes for FT-SX. The polyimide mesh was fabricated using a picosecond laser. Each hole in the polyimide mesh has irregularly shaped holes because of laser thermal damage, which may cause more crystals to settle at random orientations compared to regular shaped sample holders. A crystal sample was spread onto a polyimide-mesh, and a polyimide film was added to both sides to prevent dehydration. Using this sample holder, FT-SX was performed at synchrotron and determined the room-temperature lysozyme structure at 1.65 Å. The polyimide mesh with irregularly shaped holes will allow for expanded applications in sample delivery for FT-SX experiments.
Highlights
IntroductionThe polyimide mesh with irregular holes, nylon mesh-based sample holder, and viscous medium-based crystal supporting sample holder were exposed to an X-ray for 100 ms
Background scattering analysisThe polyimide mesh with irregular holes, nylon mesh-based sample holder, and viscous medium-based crystal supporting sample holder were exposed to an X-ray for 100 ms
Even if lasers with the same spacing and power were used to penetrate the polyimide layer, drilling resulted in irregular shaped holes because of the inherent thermal damage at the laser drilling point. We considered that this feature was very useful for enabling the random orientation of crystals on the polyimide mesh
Summary
The polyimide mesh with irregular holes, nylon mesh-based sample holder, and viscous medium-based crystal supporting sample holder were exposed to an X-ray for 100 ms. The polyimide mesh (25 μm), nylon-mesh (pore size: 60 μm), and gelatin viscous medium (thickness: < 350 μm) were enclosed in two polyimide films (total thickness: 50 μm). The X-ray energy and photon flux were 12.659 keV and 1.3 × 1 012 photons/s, respectively. 20 images were randomly extracted, and background scattering was analyzed using ADXV (https://www.scripps.edu/tainer/arvai/adxv.html). Received: 13 January 2021; Accepted: 11 June 2021
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